Plant-material sampling and grinding apparatus

ABSTRACT

Currently, no product facilitates the collection of plant material through to the production of PCR-ready DNA-template. The Plant-material Sampling and Grinding Apparatus, facilitates the collection of plant material, through to the production of PCR-ready DNA-template. The separate components of the invention include the upper and lower units. A piece of flat plant tissue is placed between the upper and lower units. Insertion of the upper unit into the lower unit cuts a disc of tissue. An extraction buffer can be introduced through the top of the upper unit and is contained by the well of the lower unit. Repeated cycles of full insertion of the upper unit into the lower unit followed by incomplete separation of the two units, facilitates the grinding of the tissue in the extraction solution. Further solutions, such as neutralizing solutions, can be added through the top of the upper unit to produce PCR-ready template.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Nil

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] Not Applicable

BACKGROUND OF INVENTION

[0003] This invention is in the general area of ‘disposable laboratoryplastic-ware.’ The invention is used for the sampling of a disc of plantmaterial (predominantly leaf tissue) and subsequent processing for thepreparation of DNA template for the Polymerase Chain Reaction (PCR).There is currently no product that facilitates the organized collectionof plant material through to the production of PCR-ready DNA-template.Currently, there are systems for the efficient production of PCR-readyDNA-extract, but none incorporate sample collection and involve thepurchase of grinding mills worth thousands of dollars.

BRIEF SUMMARY OF INVENTION

[0004] The invention, The Plant-material Sampling and GrindingApparatus, facilitates the collection of plant material, through to theproduction of PCR-ready DNA-template. The invention facilitates thesampling and processing of the tissue within the one apparatus. Theseparate components of the invention include the upper and lower units.A piece of flat plant tissue is placed between the upper and lowerunits. Insertion of the upper unit into the lower unit cuts a disc oftissue. An extraction buffer can be introduced through the top of theupper unit and is contained by the well of the lower unit. Repeatedmanipulations involving the full insertion of the upper unit into thelower unit followed by incomplete separation of the two units,facilitates the grinding of the tissue in the extraction solution.Further solutions, such as neutralizing solutions, can be added throughthe top of the upper unit for the preparation of PCR-ready template.

BRIEF DESCRIPTION OF DRAWINGS

[0005]FIG. 1: Upper (A) and Lower (B) units of the Plant MaterialSampling and Grinding Apparatus. Side view. Page 1/2.

[0006] 1) Top of upper unit possessing an opening to a cylindricalcavity.

[0007] 2) An indentation of variable shape in the top of the upper unit,for the introduction of a portion of a non-specified tool in order tosecure the upper unit. The non-specified tool will facilitate insertionof the upper unit into the lower unit and relative movements between theupper and lower units.

[0008] 3) A rib with a sharp edge to aid in the removal of excess plantmaterial.

[0009] 4) Lower cylindrical base of the upper unit that inserts into theupper cylindrical well of the lower unit.

[0010] 5) Holes of unspecified size, number, and location in the bottomof the cylindrical base of the upper unit.

[0011] 6) The bottom of the cylindrical base of the upper unit shown inconical form. Not all hidden detail is shown in order to reduce thecomplexity of drawing.

[0012] 7) A cutting edge on the bottom of the cylindrical base of theupper unit. Not all hidden detail is shown in order to reduce thecomplexity of drawing.

[0013] 8) Direction of insertion of the upper unit into the lower unit.

[0014] 9) Beveled edge on the upper cylindrical well of the lower unit.

[0015] 10) Outer wall of the upper cylindrical well of the lower unit.

[0016] 11) Inner base of the upper cylindrical well of the lower unit.

[0017] 12) Indentation to accommodate the cutting edge on the bottom ofthe cylindrical base of the upper unit.

[0018] 13) An indentation of variable shape in the middle section of thelower unit, for the introduction of a portion of a non-specified tool inorder to secure the lower unit. The non-specified tool will facilitateinsertion of the upper unit into the lower unit and relative movementsbetween the upper and lower units.

[0019] 14) Cylindrical base of the lower unit.

[0020]FIG. 2: Plan view of the Upper unit of the Plant Material Samplingand Grinding Apparatus as illustrated in figure IA except a cutting edgeon the bottom of the cylindrical base of the upper unit is not includedin this plan. Page 1/2.

[0021] 2) An indentation of variable shape in the top of the upper unit,for the introduction of a portion of a non-specified tool in order tosecure the upper unit. The non-specified tool will facilitate insertionof the upper unit into the lower unit and relative movements between theupper and lower units.

[0022] 3) A rib with a sharp edge to aid in the removal of excess plantmaterial.

[0023] 4) Lower cylindrical base of the upper unit that inserts into theupper cylindrical well of the lower unit.

[0024] 5) Holes of unspecified size, number, and location in the bottomof the cylindrical base of the upper unit.

[0025] 6) The bottom of the cylindrical base of the upper unit shown inconical form. Not all hidden detail is shown in order to reduce thecomplexity of drawing.

[0026]FIG. 3: Plan view of the Lower unit of the Plant Material Samplingand Grinding Apparatus as illustraed in FIG. 1B except an indentation toaccommodate a cutting edge on the bottom of the cylindrical base of theupper unit, is not included in this plan. Page 1/2.

[0027] 9) Beveled edge on the upper cylindrical well of the lower unit.

[0028] 10) Outer wall of the upper cylindrical well of the lower unit.

[0029] 11) Inner base of the upper cylindrical well of the lower unit.

[0030] 13) An indentation of variable shape in the middle section of thelower unit, for the introduction of a portion of a non-specified tool inorder to secure the lower unit. The non-specified tool will facilitateinsertion of the upper unit into the lower unit and relative movementsbetween the upper and lower units.

[0031] 14) Cylindrical base of the lower unit.

[0032]FIG. 4: Upper (A) and Lower (B) units of the Plant MaterialSampling and Grinding Apparatus as illustrated in FIG. 4A. Only basicfeatures are included in this figure. Side view. Page 2/2.

[0033] 1) Top of upper unit possessing an opening to a cylindricalcavity.

[0034] 2) An indentation of variable shape in the top of the upper unit,for the introduction of a portion of a non-specified tool in order tosecure the upper unit. The non-specified tool will facilitate insertionof the upper unit into the lower unit and relative movements between theupper and lower units.

[0035] 4) Lower cylindrical base of the upper unit that inserts into theupper cylindrical well of the lower unit.

[0036] 5) Holes of unspecified size, number, and location in the bottomof the cylindrical base of the upper unit.

[0037] 6) The flat bottom of the cylindrical base of the upper unit.

[0038] 8) Direction of insertion of the upper unit into the lower unit.

[0039] 10) Outer wall of the upper cylindrical well of the lower unit.

[0040] 11) Inner base of the upper cylindrical well of the lower unit.

[0041] 13) An indentation of variable shape in the middle section of thelower unit, for the introduction of a portion of a non-specified tool inorder to secure the lower unit. The non-specified tool will facilitateinsertion of the upper unit into the lower unit and relative movementsbetween the upper and lower units.

[0042] 14) Cylindrical base of the lower unit.

[0043]FIG. 5: Plan view of the Upper unit of the Plant Material Samplingand Grinding Apparatus showing basic features only. Page 2/2.

[0044] 2) An indentation of variable shape in the top of the upper unit,for the introduction of a portion of a non-specified tool in order tosecure the upper unit. The non-specified tool will facilitate insertionof the upper unit into the lower unit and relative movements between theupper and lower units.

[0045] 4) Lower cylindrical base of the upper unit that inserts into theupper cylindrical well of the lower unit.

[0046] 5) Holes of unspecified size, number, and location in the bottomof the cylindrical base of the upper unit.

[0047] 6) The flat bottom of the cylindrical base of the upper unit.

[0048]FIG. 6: Plan view of the Lower unit of the Plant Material Samplingand Grinding Apparatus showing basic features only. Page 2/2.

[0049] 10) Outer wail of the upper cylindrical well of the lower unit.

[0050] 11) Inner base of the upper cylindrical well of the lower unit.

[0051] 13) An indentation of variable shape in the middle section of thelower unit, for the introduction of a portion of a non-specified tool,in order to secure the lower unit, and facilitate relative movementsbetween the upper and lower units through the action of the nonspecified tool.

[0052] 14) Cylindrical base of the lower unit.

DETAILED DESCRIPTION OF INVENTION

[0053] There is currently no product that facilitates the organizedcollection of plant material through to the production ofPolymerase-Chain-Reaction (PCR) ready DNA template. Currently, there aresystems for the efficient production of Polymerase-Chain-Reaction (PCR)ready DNA extract, but none incorporate sample collection and involve hepurchase of grinding mills worth thousands of dollars. The systemdescribed here facilitates sampling and processing of the tissue withinthe one apparatus and does not require the purchase of expensiveequipment.

[0054] The invention is the Plant-material Sampling and GrindingApparatus. A description of the invention follows. The components of theinvention include the upper and lower units (FIGS. 1 to 6). Thedimensions and relative dimensions between sections of the invention arevariable, however further comments regarding dimensions and relativedimensions will occur throughout the detailed description when importantto unction or efficient use. The construction material is not specifiedbut is likely to be a plastic that is rigid yet possessing some elasticqualities (flexible and returns to original shape after deformation)(e.g. polypropylene).

[0055] The lower unit consists of a lower cylinder (FIGS. 1B, 3, 4B, and6; point 14), a middle section possessing an indentation suitable forinsertion of a non-specified holding and manipulating device (FIGS. 1B,3, 4B, and 6; point 13), and an upper portion that possesses an opencylindrical well (FIGS. 1B, 3, 4B, and 6; point 10).

[0056] The upper section of the Plant-material Sampling and GrindingApparatus (FIGS. 1A, 2, 4A, and 5) consists of a partially closedcylindrical base (FIGS. 1A, 2, A, and 5; point 4) and an upper sectionpossessing an indentation suitable for insertion of a non-specifiedholding and manipulating device (FIGS. 1A, 2, 4A, and 5; point 2).

[0057] The cylindrical base of the upper unit (FIGS. 1A, 2, 4A, and 5;point 4) inserts into the open cylindrical well of the lower unit (FIGS.1B, 3, 4B, and 6; point 10) as indicated in FIGS. 1 and 4 point 8. Thetightness of fit should create a seal but not so tight as to impede asmooth insertion or extraction of the upper unit.

[0058] A holding and manipulating device or grinding tool willfacilitate the insertion of the base of the upper section into the openwell of the lower section, and the extraction, or partial extraction, ofthe base of the upper section from the open well of the lower section.The tool is generally unspecified but could possess upper and lower twopronged forks that will insert into the indentations of the upper andlower sections of the Plant-material Sampling and Grinding Apparatus(FIGS. 1 to 6 points 2 and 13). The indentations could be one sided(semi-circular or possessing multiple curves); two sided (of equal orunequal sides), three sided as indicated in FIGS. 1B, 3, 4B, and 6(point 13) of equal or unequal size; or have multiple sides and angles.The forks of the tool should fit tightly enough to firmly hold the twounits and ensure appropriate alignment for insertion, but be looseenough to also allow smooth removal of the joined units from the tool.

[0059] A disc of plant tissue will be cut by placing the tissue (e.g.leaf) between the upper and lower units, while each unit is held by thetool, followed by the insertion of the upper unit into the lower unit ofthe Plant-material Sampling and Grinding Apparatus. The insertion isfacilitated by the action of the tool. Beveled edges could be includedon the upper lip of the lower unit of the Plant-material Sampling andGrinding Apparatus to create a sharp edge to improve the cutting of thesample disc (FIGS. 1B and 3 point 9). The disc of plant material will becut by the combined action of the cutting edges of the lower unit andthe downward force of the base of the upper section (FIGS. 1A, 2, 4A and5; point 6) on the tissue sample (e.g. leaf).

[0060] Grinding or extraction solutions will be introduced through theopening of the upper unit (FIGS. 1A and 4A; point 1), freely movethrough the holes in the bottom of the upper unit (FIGS. 1A, 2, 4A and5; point 5), and be contained within the upper cylindrical well (FIGS.1B, 3, 4B and 6; point 10) of the lower unit.

[0061] Complete insertion of the upper section into the lower sectiongrinds the sample between the base of the upper unit (FIGS. 1A, 2, 4A,and 5, point 6) and the base of the well of the lower unit (FIGS. 1B, 3,4B, and 6, point 11). Several cycles of partial extraction and completeinsertion would follow to complete the grinding process.

[0062] The grinding tool could have the general action of secateurs orforceps. The use of a grinding tool with secateur-like handles would beless strenuous when many samples are to be processed and allow morepressure to be applied. Options, for the grinding tool, could include;

[0063] i) Spring return handles;

[0064] ii) Pronged forks lower and parallel to main shaft to allowbetter access to Sampling and Grinding Apparatus when in a supportingplate;

[0065] iii) Multiple head tool;

[0066] iv) Locking device to ensure upper and lower sections remaintogether when grinding;

[0067] v) A tool where the focus of the arc of travel for the upper andlower components of the apparatus are behind the hand, thus reducing thecurvature of travel.

[0068] The bottom of the upper section of the Sampling and GrindingApparatus could be flat (FIGS. 4A, and 5; point 6) or otherwise, such asangled away from the sides of the cylindrical wall to form a shallowcone (FIGS. 1A and 2; point 6). When an angled and flexible bottom ofthe upper section is lowered onto a disc of leaf material positioned onthe bottom of the upper compartment of the lower section of the Samplingand Grinding Apparatus (FIGS. 1B, 3, 4B and 6; point 11), the centralregion of the cone will make contact with the tissue first. As morepressure is applied the flexible base will be pushed flat, and(increasingly) more of the tissue will be squashed by the base of theupper-section. This process will facilitate more uniform grinding andbetter mixing with the grinding solution.

[0069] A sharp protruding lip could be included on the base of the lowercylinder of the upper unit of the Sampling and Grinding Apparatus to aidin the cutting of the plant tissue (FIG. 1A point 7). The lip would fitinto a corresponding recess in the base of the well of the lower unit ofthe Plant-material Sampling and Grinding Apparatus (FIG. 1B point 12).

[0070] Sharp ribs (FIGS. 1A and 2; point 3) could be included on theupper section of the Plant-material Sampling and Grinding Apparatus, tohelp in the removal of excess plant material.

[0071] The suggested method of use is represented by the followingpoints;

[0072] i) 96 Plant-material Sampling and Grinding Apparatus (uppersection inserted into lower section) designed to fit into the wells ofone or more commercially available, standard, 96-well microtiter-plates(apparatus supported by the insertion of the lower cylindrical base ofthe lower unit (FIGS. 1B, 3, 4B and 6; point 14) into a well of theplate, the base of the lower unit should fit tightly into the wells butnot so tight as to impede a smooth extraction, individual apparatusshould not be touching, and most likely constructed of a rigid, yetpossessing some elastic qualities, (flexible and returns to originalshape after deformation) plastic (e.g. polypropylene)).

[0073] ii) 96 Plant-material Sampling and Grinding units (upper sectioninserted into the lower section) purchased pre-packaged and supported ina 96 well sampling plate (not a standard 96 well microtiter plate) bythe insertion of the lower cylindrical base of the lower unit (FIGS. 1B,3, 4B and 6; point 14) into a well of the sampling plate. The base ofthe lower unit should fit tightly into the wells but not so tight as toimpede a smooth extraction. The wells of the sampling plate could besufficiently separated to allow easy access of the grinding tool to theSampling and Grinding units. The sampling plate will have a lid. Theplate with lid could slot onto a lower container. The three componentswould constitute the Sampling Container. The lower container could befilled with ice or a cold freezer block thus facilitating reduced sampledeterioration. Alternatively, the Plant-material Sampling and Grindingunits (upper section inserted into the lower section) could be suppliedloose or in cassettes that could be stored in a holding rack forsubsequent loading of the Sampling and Grinding units into the SamplingPlate and subsequent utilization of the Sampling Container.

[0074] iii) Take labeled Sampling and Grinding units, contained withinthe cooled Sampling Container, to the experimental plants. Sample leafdiscs, and return apparatus to the wells of the sampling container.Return to the laboratory.

[0075] iv) An adaptor will be used to facilitate the addition, bymulti-channel pipettor, of, for example, 200 micro liters of extractionsolution (e.g. 0.25 N NaOH), into each Sampling and Grinding Apparatus.The adaptor will be placed on top of the sampling tray containing 96apparatus. Tubes or tunnels passing through the adapter will insert intothe opening in the upper unit (FIGS. 1A and 4A; point 1). The tubes ortunnels will lead from the openings in the top of the adapter to theapparatus in the sampling plate, maintaining spatial relationships. Theopenings will have the configuration of a standard 96-wellmicrotiter-plate. Remove adaptor.

[0076] v) Each leaf sample will then be ground, with the aid of thegrinding tool, until the solution has turned to a pale green color. Thenumber of units ground simultaneously will depend on the number ofholding forks on the head of the grinding tool.

[0077] vi) After grinding, Plant-material Sampling and Grinding unitscould then be transferred to a standard, commercially available, 96-wellmicrotiter-plate, supported by the cylindrical base of the lower unit(FIGS. 1B, 3, 4B and 6; point 14) within the wells of the plate.

[0078] vii) 50 micro liters, for example, of extract solution would thenbe removed from each unit and placed into the well of a standard,commercially available, 96 well microtiter plate that already contains,for example, 200 micro liters of neutralization buffer (e.g Tris bufferpH 7). Multi channel pipettors should be used if available.

[0079] viii) 2 micro liters of the neutralized extract could be used astemplate for a 20 micro liter PCR amplification.

[0080] ix) Alternatively, continuing from step 5, neutralizationsolution could be added individually to the original apparatus, eitherdirectly, or indirectly through the adaptor. It this case, PCR readytemplate would be produced in the original sampling apparatus.

What I claim as my invention; 1) the plant-material sampling andgrinding apparatus, consisting of an upper and lower unit, substantiallyas herein described with reference to the accompanying drawings; thatwill facilitate the cutting of a disc of flat plant material by placingthe tissue between the upper and lower units, while each unit is held bya tool, followed by the insertion of the upper unit into the lower unit,facilitated by the action of the tool, thus containing the sampledtissue in the well of the lower unit; that will permit the addition ofsolutions to, and extraction of solutions from, the area containing thesampled material, through the opening in the top of the upper unit; thatwill facilitate the grinding of the sampled material with addedsolutions, by repeated cycles of complete insertion of the upper unitfollowed by partial extraction of the upper unit from the lower unit;that will facilitate the production of Polymerase-Chain-Reaction readyDNA-template: a. The Plant-Material sampling and Grinding Apparatus fromclaim 1 designed so that 96 Apparatus (upper section inserted into lowersection) are supported by the wells of one or more, commerciallyavailable, standard 96-well microtiter-plates (apparatus supported bythe insertion of the lower cylindrical base of the lower unit into awell of the plate), the base of the lower unit should fit tightly intothe wells but not so tight as to impede a smooth extraction, andindividual apparatus in contact or not in contact with each other whensupported in the microtiter plate. b. The Plant-Material sampling andGrinding Apparatus from claim 1 constructed of a rigid, yet possessingsome elastic qualities, (flexible and returns to original shape afterdeformation) plastic (e.g. polypropylene)).